Such methods are employed in medical technology, biotechnology and in pharmaceutical technology, e.g. to detect antigen-antibody reactions.
An immobilization layer is defined as a coating of atoms or molecules that can selectively bind to other molecules. Interactions are defined as interactions between the atoms or molecules that are anchored to the immobilization layer and the molecules to be bound. These can be biological or chemical reactions.
Biological and chemical reactions that take place in liquid-filled cuvettes while forming thin films have thus far been detected, inter alia, by marking the substances involved, e.g. by fluorescent or radioactive molecules. This is described in S. S. Deshpande, “Enzyme Immunoassays—From Concept to Product Development,” Chapman & Hall, 1996. This method is relatively simple to perform but has a number of drawbacks. The relevant molecules must first be labeled or purchased in labeled form. While these preparations are time-consuming, marking can moreover influence the biological or chemical interactions, which in turn affects the measurement results. The problems inherent in handling radioactive materials are a further drawback.
For this reason direct measuring methods that require no marking at all are increasingly being used. Two methods have proven suitable.
In surface plasmon resonance measurement, the resonance of free electrons is excited in approximately 50-60 nm thick metal layers, particularly gold or silver, (see B. Gedig, D. Trau and M. Orban, “Echtzeitanalyse biomolekularer Wechselwirkungen”, [Real Time Analysis of Biomolecular Interactions] Laborpraxis, February 1998, pp. 26-28 and 30). This excitation of the free electrons occurs only if polarized light is applied parallel to the plane of incidence. For each measurement, either the angle of incidence or the employed light frequency must be passed through, so that the instrumentation is relatively complex. The reflected intensity as a function of the wavelength at a fixed angle or of the angle of incidence at a fixed wavelength shows a minimum in the resonance range.
Since the electromagnetic radiation when reflected is not confined to the thin metal film but in the so-called evanescent field interacts with approximately the first 100 to 300 nm of the medium above this film, the resonance angle or the resonance wavelength strongly depends on the refractive index of the layer located directly above the metal film. If the resonance conditions change, for instance, because small amounts of water are being replaced through biological or chemical reactions with formation of an additional layer, the minimum of the reflected intensity is shifted. This shift makes it possible only qualitatively to detect the growth of the layer but not its absolute thickness, for which the refractive index of the growing layer would have to be known. Thus, despite the substantial complexity of the instrumentation, the measurement result is not very meaningful. A corresponding measuring apparatus is described, for instance, in WO 90/05295.
The second method is ellipsometry in which the light is applied in such a way that it passes through a gaseous or liquid ambient medium and subsequently strikes the biological or chemical layer to be detected (see H. Arwin, “Spectroscopic ellipsometry and biology: recent developments and challenges,” Thin Solid Films 313-314, 1998, pp. 764-774). In ellipsometric measurements, the ellipsometric parameters Ψ and Δ are determined, for which the following is true:rp/rs=(Erp/Eep)/(Ers/Ees)=tan Ψ·exp (iΔ)rp, rs: complex reflectivities                E: complex electric field amplitude        Indices: p: parallel to the plane of incidence                    s: perpendicular to the plane of incidence            e: radiated            r: reflected                        
Ψ essentially includes the change in intensity through reflection of the light. Δ essentially includes the phase shift through reflection of the light; this parameter reacts very sensitively to film thicknesses.
EP 0 067 921 describes a biological test method for determining bioactive substances by means of ellipsometric measurements. A thin dielectric substrate is coated with an immobilization layer of a first biologically active substance, which interacts with a second bioactive substance. The optical changes in the biological layer are detected by ellipsometric measurements. Analysis is effected by plotting the ellipsometric parameters as a function of time and comparing these curves with reference curves obtained by measuring biological material with known concentrations. Irradiation through the back of the substrate was taken into consideration, but the sensitivity of the measurement with irradiation from the back was 30× poorer than with irradiation from the front. As a result, this known method has the drawback that special cuvettes are required and titer plates cannot be used at all.
In Sensors and Actuators B 30 (1996), pp. 77-80, it is proposed to examine the polarization state of the reflected light to detect DNA samples that are immobilized on a metal layer. As a reference, a metal layer without DNA molecules is examined. Both p- and s-polarized light is applied and the phase shift between the samples and the reference signal is evaluated. Instead of examining the angular dependence of the intensity as in the known surface plasmon measurements, the angular dependence of the polarization state is examined here.
Implementation in practice, however, would again require a complex apparatus due to the changes in the angle of irradiation that have to be made.